Improving the measurement of nitrogen stable isotopes in organic materials containing high C:N ratios using a 5A molecular sieve column

The nitrogen stable isotope composition (δ15N) of plant materials has numerous applications. Plant materials like bark can have a very high C:N ratio. Incomplete C combustion in such samples interferes with the δ15N measurement due to CO production. We modified the standard setup for δ15N measurement using an elemental analyzer (EA) coupled to an isotope ratio mass spectrometer (IRMS) by incorporating a 5A molecular sieve column, which better separates N2 from CO. We compared this new modified setup and the standard one for the measurement of bark samples. Precision and accuracy for δ15N in standards with low C:N ratio were equivalent for the two methods. However, for bark the results obtained with the new method had better precision and accuracy than the standard method. Replicates are nevertheless recommended with the new method to ensure confidence in the results.• During elemental analysis, incomplete combustion of material with high C:N ratio can lead to CO formation, which interferes with δ15N IRMS measurements.• Here we use a 5A molsieve column to remove the CO interference in δ15N measurements Precision and accuracy on δ15N measurements of samples with high C content are significantly improved

• During elemental analysis, incomplete combustion of material with high C:N ratio can lead to CO formation, which interferes with  15 N IRMS measurements.• Here we use a 5A molsieve column to remove the CO interference in  15 N measurements Precision and accuracy on  15 N measurements of samples with high C content are significantly improved

Specifications table
Subject area: Chemistry More specific subject area: Stable isotope measurement Name of your method: 5A molsieve for  15 N Name and reference of original method: Not applicable Resource availability: Not applicable

Background
In our laboratory, we have been employing the standard method to measure  15 N [ 1 ] for almost two decades.In this method, a sample of a solid powder is wrapped using tin foil, placed in an autosampler [ 2 ], and then combusted at 1020 °C in a combustion reactor.A pulse of oxygen is added at the time of the combustion to ensure a complete reaction, converting all carbon to CO 2 , and all nitrogen to NO or NO 2 .Helium flows constantly and carries the gases to a reduction reactor at 650 °C, where all nitrogen is reduced to N 2 .The He stream carries the resulting gases through a water trap to remove any traces of water, and a then through a Gas Chromatography (GC) column kept at 40 °C, where N 2 and CO 2 are separated from each other and from other potential trace gases, such as O 2 which is used to aid combustion.The GC column used here is proprietary, that is, the manufacturer (Sercon) did not disclose its composition.However, such GC columns are usually made using Hayesep Q or Hayesep T meshes [ 3 ].The gases are finally carried to an isotope ratio mass spectrometer (IRMS) where their stable isotope composition (  15 N for N, and  13 C for C) are measured.Stable isotope measurements are reported using the "delta notation ", in which the measured values are compared to internationally agreed reference values; the SI unit for values using the delta notation is Urey, usually expressed as milli Urey, or mUr.If only 15N is necessary, a CO 2 trap (e.g., soda lime) can be placed before the water trap, allowing for a shorter analysis time.
The method described above is robust and works for a large majority of organic samples.However, the measurement of  15 N in tree bark has been found to be problematic due to its high C:N ratio, which demands relatively large samples to be used in order to achieve higher precision in  15 N measurements.Nevertheless, this leads to an incomplete combustion of the C in the sample, and to the production of CO instead of CO 2 [4][5][6][7].The CO 2 trap does not work for CO, and the GC column does not separate CO from N 2 with the same efficiency as it does for CO 2 .Consequently, a CO peak comes very close to the N 2 peak, affecting the calculated  15 N values ( Fig. 1 A).
In some cases, however, visual inspection of the chromatogram does not always allow one to rule out CO interference on  15 N ( Fig. 1 B).

Method details
Here we follow the standard method described above, with a small modification.When analyzing bark samples in our laboratory we replace the standard GC column (SC1996, by Sercon) with a 5A molsieve column (SC8428, by Sercon), which is known to better separate CO from N 2 .This way, even if a very small CO peak forms, it is separated from the N 2 peak, and 15N can be measured without interference ( Fig. 2 ).The 5A molsieve column is kept at 40 °C during measurements.

Example 1: Bark samples
The standard method was compared to the modified method for  15 N measurements using the bark of the wetland tree species Melaleuca quinquenervia .Bark samples where oven-dried at 60 °C for 4 days and subsequently homogenized by adding stainless steel beads (Qiagen) and employing a TissueLyser II (Qiagen) at 26 cycles/ sec for one minute or more until finely powdered.In both cases, measurements were conducted for finely powdered bark samples ( ∼40 mg each), and measured alongside in-house standards ( ∼1 mg each) with known  15 N composition (glycine:  15 N = 2.0 mUr and caffeine:  15 N = − 4.2 mUr), which had been determined by measuring them previously against international standards (USGS64:  15 N = 1.8 mUr and USGS65:  15 N = 20.7 mUr).Samples and standards were prepared using an autosampler [ 8 ], with amounts targeted to obtain peaks large enough to minimize blank influence (usually < 0.1 mUr).Measurements were conducted under identical conditions (oxidation reactor at 1020 °C, reduction reactor at 650 °C, soda lime to trap CO 2 , magnesium perchlorate to trap H 2 O, 30 s of O 2 injection, measurement cycle lasting 800 s), except for the GC column used.For the standard method the SC1996 (by Sercon) GC column was utilized, while for the modified method the Fig. 2. Chromatogram for the measurement of a bark sample performed using the modified setup, where a CO peak is present together with a N2 peak without interference.The measurement produced the expected  15 N value of 0.5 mUr.

Table 1
Comparison between the standard and modified methods for the measurement of  15 N (unit: mUr; values shown as average ± standard deviation, followed by the number of measurements) in bark samples.P values are for unpaired Student t Test for means on a same table row.

Substance
Standard method Modified method P value Glycine (working standard) 2.0 ± 0.08 n = 6 1.9 ± 0.13 n = 7 0.39 Caffeine (working standard) SC8428 (by Sercon) GC column was used.Both columns were kept at 40 °C.In both treatments the samples were intercalated with empty cells in the autosampler, because previous experiments showed that consecutive bark measurements using SC8428 became unstable (results not shown).
Results for working standards were consistent for both methods ( Table 1 ), indicating that both methods provided accurate and precise results for those substances.In contrast, results for bark samples obtained with the standard and modified methods presented significant differences ( Table 1 , p < 0.001), indicating that one (or both) of the methods was inaccurate.The standard method exhibited poor precision, and  15 N values could be very high (e.g., 104.6 mUr) when CO interference was strong, indicating its inaccuracy.
Precision for bark samples using the modified method was notably improved ( Table 1 ), and after removing four potential outliers, it matched the precision of the standards (0.4 ± 0.15 mUr, compare to Table 1 ).The three outliers had very similar values (0.0 ± 0.08 mUr), suggesting that they are not the result of poor homogenization, but rather some unaccounted-for uncertainty in the analytical procedure.Therefore, it is recommended that samples measured using this method are replicated, to account for potential less-thanoptimum precision.
The accuracy of the modified method is supported by the improved precision for bark samples, and the precision and accuracy for the working standards ( Table 1 ).Furthermore, the obtained average value for bark (0.5 mUr) is a common value for terrestrial plants [ 9 ].

Example 2: Evaluating accuracy
The experiment with bark samples demonstrated that the modified method generated acceptable precision ( Table 1 ) for both standards and bark samples.However, since the  15 N of the bark samples was initially unknown, the only evidence for its accuracy is that the obtained value is a common value with an acceptable precision.In order to explore possible deviations from accuracy with the modified method, mixtures of substances with known  15 N, such as cellulose, were prepared and measured.The proportions of the mixtures were determined to mimic the C:N ratio ( ∼300) of the bark.Only the modified method was used this time.
Although cellulose was expected to be N free, trace amounts of N were observed in it, which made necessary a blank correction for  15 N in the measurements using a mass balance.The  15 N in cellulose was found to be 14.6 ± 1.87 mUr ( Table 2 ).The low precision was due to the small peak size.This value was used to obtain corrected  15 N values for the mixtures.The precision of the mixture measurements was lower than for pure substances due to this correction ( Table 2 ), especially for caffeine.However, the average values for unmixed and mixed substances were very similar ( Table 2 ), suggesting that the modified method yields accurate results.Significant values are marked with an * (  = 0.05).

Limitations
The 5A column is useful for  1 7 N measurements, but care should be taken if the CO 2 trap becomes exhausted.If so, the 5A column can trap CO 2 until its saturation, but then the column releases any new CO 2 , making it impossible to do any measurement.Therefore, the column must be periodically baked between measurements to ensure that CO 2 or other impurities are expelled.In our experience, baking the column at 190 °C for 2 h after every 100 measurements worked well.Also, the 5A column cannot be used for  1 tC measurements, as it traps CO 2 at 40 °C.
In summary, the application of the 5A molsieve column was successful for measuring  15 N in bark samples, and should work with other samples with a high C:N ratio.However, other analytical strategies could potentially achieve similar results.For example, the elemental analyzer commercialized by Elementar for  15 N uses a different analytical principle from that used here [ 10 ].It is possible that their standard setup does not suffer from CO interference in the same way as described here.

Fig. 1 .
Fig. 1.Chromatograms illustrating measurements of bark samples conducted using the standard setup, showing the presence of a CO peak alongside a N 2 peak.A) The CO peak is clearly separated from the N 2 peak.B) The CO peak is less distinct, although likely present.Despite the measured sample having a known  15 N value of 0.5mUr, the calculated values for Fig. 1 A and Fig. 1 B were 25.0 mUr and 7.6 mUr, respectively.

Table 2
Measurement of  15 N in substances with known  15 N using the modified method.Data shown as in Table1, except for P values that refer to unpaired Student t-test for averages between unmixed and mixed substances (glycine versus glycine plus cellulose; caffeine versus caffeine plus cellulose).